Recently, an ideal therapeutic method for cancer, with less side effects, has been desired, wherein normal cells are not affected and only the cancer cells can be selectively impaired. For example, gene therapy is capable of increasing the selectivity of the cancer cell at various levels, such as the cell selectivity and expression promoter activity of a gene to be introduced into the cancer cell, or infection and induction method of a viral vector. However, there is a common problem. Since expression of a tissue-specific differentiation antigen in the immunotherapy for cancer is also observed to some extent in normal cells, the side effects to the normal cells pose a problem. Furthermore, since cancer antigen with a mutation is limited to the individual cancers, it is not suitable as an immunotherapy for cancer that is molecule-targeted.
Recently, a clinical study of gene therapy was conducted to treat a malignant brain tumor using a replication-competent herpes simplex virus (HSV) (vector) that continuously and selectively impairs only the proliferating cells by infection and replication (Gene Ther. 7, 859-866, 2000; Gene Ther. 7, 867-874, 2000). The replicative HSV vector is a vector with deleted Ribonucleotide reductase (RR) or Thymidine kinase (TK) that are essential for viral replication. These enzymes express in normal cells only when they are proliferating but express constitutively in tumor cells. Therefore, when this HSV vector infects a cell that proliferates strongly, regardless whether it is a normal cell or a tumor cell, it replicates with cell-derived RR or TK and shows a cytolytic activity. Meanwhile, an anti-tumor effect of replicative HSV vectors against prostate cancer and pancreatic cancer has also been reported in an animal experiment (J. Surg. Oncol. 72, 136-141, 1999), however, these vectors do not have cell selectivity either, and their safety is low. Therefore, while such a vector could be used in therapy of human brain tumors, since this vector does not diffuse into the circulating blood due to the presence of a blood brain barrier, it is not suitable for treatment in any other organs.
Thus, a further effective and safer therapy can be implemented if the impairment activity of the HSV vector is controlled target in cell-specifically. Martuza et al. reported on a replication-competent HSV vector that is liver tumor-selective, using an albumin promoter (J. Virol. 71, 5124-5132, 1997). However, when this vector is used in liver cell cancer, the expression of albumin gene decreases and the normal regenerative liver cells are also impaired. Therefore, it is not considered suitable for clinical application in human. The description of U.S. Pat. No. 5,728,379 (“Tumor- or cell-specific herpes simplex virus replication”) mentions the possibility of application of this system to mesothelioma, however, it does not state the possibility of application to therapies for human sarcoma in general, such as leiomyosarcoma, osteosarcoma, gastrointestinal stromal tumor (GIST), tumor vessel, proliferating vascular lesion, proliferating glomerulonephritis, fibrosis of lung, liver and the like, or myofibroblast that proliferate at the stroma of malignant tumors.
The existence of fusion gene and mutation of p53 and Rb in some tumors are reported based on the genetic analysis of the disease cause and pathology of sarcoma, with yet limited applicability to therapies. In an animal experiments using nude mice, Milas et al. employed an adenoviral vector without a replication ability to introduce p53 gene into leiomyosarcoma cells, and reported that a delayed effect in the proliferation of tumors (Cancer Gene Ther. 7, 422-429, 2000). A method for introducing and expressing a suicide gene, thymidine kinase, into osteosarcoma by using an osteocalcin gene promoter has also being described (Cancer Gene Ther. 5, 274-280, 1998). However, it uses a viral vector with no replication ability which results in the poor efficiency for gene transfer Such system, therefore, cannot be applied to sarcoma other than osteosarcoma. Particularly, according to Milas et al., a human smooth muscle cell line (SK-LMS-1) were infected with 100 to 1000 fold more amount of viral particles with lower efficiency in comparison to the amount of particles of the viral vector used in the report (Cancer Res. 61, 3969-3977, 2001) by the present inventors. Therefore, the approach of Milas et al. is not preferable, from the viewpoint of suppressing the side effects by minimizing the number of viral particles to be injected into the body.
Furthermore, Folkman, et al. reported a dramatic anti-tumor effect of anti-angiogenesis peptides such as angiostatin and endostatin in mice as a therapy for suppressing angiogenesis of cancer (Cell 79, 315-328, 1994; Cell 88, 277-285, 1997). Nakamura et al. also reported the suppressing action of angiogenesis of NK4, an intramolecular fragment of a hepatocyte growth factor (HGF) (Biochem. Biophys. Res. Commun. 279, 846-852, 2000). However, these methods have problems, such as (1) the requirement for a large amount of peptides, (2) their reproducibility to endostatin is low, (3) the mechanism is unknown, and (4) the efficacy in human has not been confirmed. The inhibitor of angiogenesis, which is currently in clinical trial, does not have cell selectivity and its inhibiting efficiency is low. The peptide which inhibits the action of the integrin on the surface of endothelial cells, reported by Cheresh et al., does not have cell selectivity as well, and its inhibiting efficiency is low (J. Clin. Invest. 103, 1227-1230, 1999). These studies all relate to therapies that target vascular endothelial cells, however, cell-selective therapeutic agent targeting tumor vessel composed of proliferating vascular smooth muscle cells has not been known. In fact, it is reported that the antagonist of a platelet-derived growth factor receptor that facilitates the proliferation and migration of smooth muscle cells has a strong suppressing action for tumor angiogenesis (Cancer Res. 60, 4152-4160, 2000), and the importance to attack the vascular smooth muscle in order to suppress the tumor angiogenesis is speculated. However, this method is not cell-selective and side effects are also expected.
Moreover, various agents that suppress the proliferation of smooth muscles of neointima have been examined for the proliferating vascular lesion, in particular, vessel constriction after stent placement and heart transplantation. However, none of these agents have succeeded in preventing constriction. As a recent attempt in gene therapy, there is a report by Leiden et al., to selectively introduce a LacZ gene into a smooth muscle cells of a rat carotid artery after balloon injury, under the control of a promoter of SM22a, a homologous gene of calponin using an adenoviral vector that is deficient in replication ability (J. Clin. Investi. 100, 1006-1014, 1997). However, in this experiment, it was not the proliferating smooth muscle of the intima that is a target cell, but the smooth muscle of the tunica media that was introduced with the LacZ gene, and the efficiency of introduction was extremely low. Further, Nabel et al. also conducted an experiment using an adenoviral vector without a replication ability, wherein a LacZ gene and CAT (chloramphenicol acetyltransferase) gene were introduced into pig artery under the control of SM22a prompoter. However, only 2.2% of the intimal smooth muscle cells, 0.56% of the tunica media smooth muscle cells showed gene expression (Mol. Med. 6, 983-991, 2000). In contrast, according to Miyatake et al., the replication of HSV virus, which was used to infect rat carotid artery after balloon injury, is observed mainly in the proliferating smooth muscles of the intima, and the efficacy of using a replicative viral vector is speculated (Stroke 30, 2431-2439, 1999). However, this virus is not cell-selective and side effects such as the cell disruption of intima cells and adventitial fibroblasts are expected. Other methods such as directly introducing decoy and antisense DNA oligonucleotide into the vessel have also been attempted, however, the efficiency of introduction is low and sufficient suppressive effect of vessel smooth muscle proliferation is unlikely.
In another recent attempt of gene therapy based on proliferating mesangial cells in glomerulonephritis, a method has been reported wherein decorin and TGFB receptor are introduced into the renal glomerulus using a liposome vector. The receptors inhibit TGFβ1 and chimeric gene of the IgG Fc region, or decoy of NFkappaB (Nature Med. 2, 418-423, 1996; Kidney Int. 55, 465-475, 1999; Gene Ther. 7, 1326-1332, 2000). However, this method is not cell-selective and side effects are also expected. Moreover, a method has been presented, wherein an adenoviral vector deficient in replication ability is bound to a microsphere of polystyrene and administered to a rat aorta, in order to selectively introduce a gene into a renal glomerulus (Kidney Int. 58, 1500-1510, 2000). However, aside from mesangial cells, which are a cause for proliferating glomerulonephritis, expression of introduced genes is observed also in vascular endothelial cells. Further, the immunogenicity of adenovirus is strong, and there is a high risk for it to evoke the immune response that leads to glomerulonephritis (Kidney Int. 61, S85-S88, 1997).
Meanwhile, the present inventors have found that a calponin gene, which is thought to be a differentiation marker of smooth muscles, is expressed in the tumor cells of human-derived sarcoma (Int. J. Cancer 79, 245-250, 1998; Sarcoma 3, 107-113, 1999; Intern. J. Cancer 82, 678-686, 1999). Thereafter, there have been continuous reports that calponin genes express abnormally in almost 20 types of human malignant tumor derived from mesenchymal cells such as bone sarcoma and soft tissue sarcoma as well as in gastrointestinal stromal tumor (GIST) and salivary gland sarcoma, fibrosarcoma, malignant neurinoma. The X-ray crystallographic structure and the in vitro and in vivo functional analyses of the calponin (h1 or basic) revealed that calponin binds to the C-terminal region of actin molecules and suppresses the sliding motility of actin and myosin (Biochem. Biophys. Res. Commun. 279, 150-157, 2000; J. Physiol. 529, 811-824, 2000). In an adult body, the calponin gene selectively expresses in the smooth muscle cell and is regarded as a differentiation marker of the vessels and gastrointestinal tract (Physiol. Rev. 75, 487-517, 1995).
U.S. Pat. No. 5,728,379 mentioned above and the report by the present inventors (Cancer Res. 61, 3969-3977, 2001) further describe a replicative vector deficient in a DNA that encodes a thymidine kinase of HSV. However, HSV deficient in thymidine kinase is not sensitive to aciclovir or ganciclovir, which are anti-herpes virus agents, and when these vectors are applied in therapies for human, there would be serious safety concerns if the expansion of unexpected infection of the virus occurs.
In a recent study a replication-competent HSV-1, G207, has been prepared. The vector is deficient in both copies of the gamma 34.5 gene that are involved in the replication in the neuronal cells, has the LacZ gene inserted in the ribonucleotide reductase (ICP6)-locus (Nature Med. 1, 938-943, 1995). Another replication-competent HSV-1 vector HSV1yCD has an autofluorescent protein and a cytosine deaminase that are expressed by a CMV promoter/enhancer inserted in the ICP6-locus by homologous recombination (Cancer Res. 61, 5447-5452, 2001). However, since both are deficient in ribonucleotide reductase, it is anticipated that both vectors would replicate in proliferating cells alone but there would be no cell selectivity. Moreover, there is no report of a treatment method wherein the proliferating myofibroblast in the fibrosis such as pulmonary fibrosis and hepatic fibrosis is targeted and selectively disrupted. In addition, there have been no report of a treatment method wherein the myofibroblast that proliferate of malignant tumors are targeted.